Accelerator of the activity of hydrolase

ABSTRACT

An accelerator of the activity of hydrolase consisting of a coordination compound or a metal complex containing, as a ligand, an organic cyclic compound with carbonyl groups adjacent to each other in the structure shown in the following general formula I that are substantially in the same plane as each other; ##STR1## (wherein A is the cyclic compound moiety.)

BACKGROUND OF THE INVENTION

1. Field of the Invention:

This invention relates to an accelerator of the activity of hydrolase,especially one that accelerates the activation of the precursors ofserine protease, including blood coagulation Factor XII, and thataccelerates the enzyme activity of serine protease produced by theactivation of the said precursors. Moreover, this invention relates toan accelerator of blood coagulation with the use of the saidaccelerator.

2. Description of the Prior Art:

The remarkable progress in testing techniques coupled with the spreadingof blood tests including serum biochemistry tests, serum immunologytests, and blood cell tests is contributing much to the prevention ofdisease and to early diagnosis. Most blood tests are tests of the serum,and the serum that is needed for such tests is, as a rule, obtained byblood that is collected into a container for blood tests and allowed tocoagulate, after which centrifugation takes place, by which the bloodclot (the gel-like mass of a mixture of fibrin and blood cells), whichhas a different specific gravity from that of the serum, is separatedtherefrom, and the serum is collected with the use of a pipette or bydecantation.

A relatively long period of time is needed for blood collected fromsubjects to be tested to coagulate. For example, even if glass testvessels, which allow blood to coagulate in a relatively short period oftime, are used, it takes 40-60 minutes until coagulation, and if testvessels made of a synthetic resin are used, the vessels must be left for4 hours or more. For that reason, there is the problem that it is notpossible to obtain serum needed for tests speedily. This is a particularproblem when the test is to be done in a emergency.

A blood coagulation accelerator is used so that blood will coagulaterapidly. Factor XII is one form of precursor of a protein hydrolase(serine protease) related to blood coagulation, and by its activation,other blood coagulation factors in the blood are activated in turn tostart blood coagulation. As accelerators of the activity of Factor XII,conventionally, glass, kaolin, bentonite, silica, and other inorganicfine or coloidal particles, and ellagic acid are known. These are alsoused as one ingredient among the reagents for the measurement of theactivated partial thromboplastin time (APTT), which is one kind of testof coagulation functioning. However, even if these are used asblood-coagulation accelerators, differences in their purity andcomposition cause scattering of the results for tests such as the timefor blood coagulation. In addition, even if these accelerators of bloodcoagulation are used to promote coagulation of the blood, the separationeffect of the blood serum and the blood clot is incomplete, so whencentrifugation is done to obtain the serum, some components of the bloodclot are mixed into the serum, which is a disadvantage.

A further problem arises when serum is obtained from the blood ofpatients who receive hemodialysis or patients with blood-clottingdisorders. Such patients are given heparin to prevent the formation ofclots, so 1-20 units of heparin is found in every 10 ml of their blood.This heparin binds with antithrombin III in their blood and stronglyinhibits the effects of thrombin. It is said that the heparin alsoinhibits the effects of blood coagulation factors including Factor XII.For that reason, fibrinogen does not become fibrin, and so blood doesnot coagulate. Even if the accelerators of blood coagulation mentionedabove are added, the blood does not actually coagulate, and so it isdifficult to obtain the serum.

The inventors of this invention have proposed in Japanese Laid OpenPatent Publication No. 60-115519 that an organic cyclic compound withthe following general formula has carbonyl groups adjacent to each otherin the structure that are substantially in the same plane as each otherand is useful for accelerators of the activity of hydrolase, which canbe used for accelerators of blood coagulation. ##STR2## (wherein A isthe cyclic compound moiety.)

As above mentioned compounds, for example, there are oxidized alkylgallate, oxidized ellagic acid, etc. These compounds non-enzymaticallyactivate blood coagulation Factor XII, and make it possible for theblood to coagulate in a relatively short period of time. The effect isstronger than that obtained with the use of conventional accelerators ofblood coagulation. The inventors have also proposed an accelerator ofblood coagulation in which these compounds are the main ingredient, orin which various additives are added to these compounds. For example,applications have already been filed for an accelerator of bloodcoagulation that contains protein hydrolase as a co-accelerator topromote further the coagulation of the blood (Japanese Laid Open PatentPublication No. 60-174952) and for an accelerator of blood coagulationthat includes a neutralizer of heparin so that the heparin in the bloodof patients receiving heparin will be neutralized (Japanese Laid OpenPatent Publication No. 60-27858).

It is possible to use all kinds of proteases including trypsin,thrombin, and cathepsin B as protein hydrolases for the acceleration ofblood coagulation. These protein hydrolases act to accelerate theactivation of blood coagulation factors in the blood, so the bloodcoagulates rapidly. For the neutralizer of heparin mentioned above,compounds with amine salts and/or a quaternary nitrogen can be used,including alkylamine hydrochloride. With an accelerator of bloodcoagulation that includes such compounds, the above-mentioned aminesalts, etc., adsorb onto heparin, neutralizing it, and act to inactivatethe heparin; moreover, the compounds with the structure of formula Iactivate blood coagulation Factor XII in the blood, so that it becomespossible for the blood to coagulate in a short period of time.

As mentioned above, by the use of an accelerator of blood coagulationcontaining compounds of the structure shown in formula I as the mainingredient, it is now possible to obtain serum from blood that hascoagulated in a short period of time, having been taken from eitherhealthy persons or patients given heparin. There is not a largescattering in the time taken for the blood to coagulate. However, thecompounds of the structure shown in formula I mutually interact withsome components of blood and influence the data obtained by clinicalexaminations, so that the use of the compounds as the main ingredient ofan accelerator of blood coagulation must be limited.

SUMMARY OF THE INVENTION

The accelerator of the activity of hydrolase of this invention, whichovercomes the above-discussed and numerous other disadvantages anddeficiencies of the prior art, consists of a coordination compound,namely a metal complex containing, as a ligand, an organic cycliccompound with carbonyl groups adjacent to each other in the structureshown in the following general formula I that are substantially in thesame plane as each other; ##STR3## (wherein A is the cyclic compoundmoiety.)

In a preferred embodiment, the hydrolase is serine protease.

In a preferred embodiment, the hydrolase is blood coagulation FactorXII.

The accelerator of blood coagulation of this invention contains theabove-mentioned accelerator of the hydrolase activity.

In a preferred embodiment, the accelerator of blood coagulation furthercontains hydrolase as a co-accelerator, the hydrolase being an enzymethat can hydrolyze the bond between Arg and any amino acid residueand/or the bond between Lys and any amino acid residue on a peptidechain.

In a preferred embodiment, the hydrolase contained in the saidacceleration of blood coagulation is at least one selected from serineprotease, thiol protease, and metal protease.

In a preferred embodiment, the accelerator of blood coagulation furtherincludes an organic compound that has an amino salt and/or a quaternarynitrogen.

In a preferred embodiment, the accelerator of blood coagulation furtherincludes an anti-fibrinolysis agent and/or an anti-plasmin agent.

For these reasons, the invention disclosed herein makes possible theobjects of (1) providing an accelerator of the activity of hydrolasethat can be an excellent accelerator of blood coagulation; (2) providingan accelerator of the activity of hydrolase as mentioned above that isreadily manufactured and refined; (3) providing an accelerator of theactivity of hydrolase that can be stored for long periods of timebecause it is relatively stable against heat; (4) providing anaccelerator of blood coagulation that includes the accelerator of thehydrolase activity and promotes the coagulation of blood in a shortperiod of time; (5) providing an accelerator of blood coagulation thatgives good separation effect of the serum and the blood clot andseparates the serum from the blood clot in a high yield; (6) providingan accelerator of blood coagulation that does not change the serum, sothe serum can be used for every kind of biochemical test and clinicaltest to give accurate results at all times with reliability; (7)providing an accelerator of blood coagulation that promotes the speedycoagulation of blood even if the blood contains heparin, and with whichthere is stability even after coagulation, and also good separation ofserum.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compounds shown in formula I below, which are a ligand of thecoordinate compound, namely a metal complex (i.e., the accelerator ofthe activity of hydrolase of this invention), can be homocycliccompounds or heterocyclic compounds; also, they can be monocyclic orpolycyclic compounds. ##STR4## (wherein A is the cyclic compoundmoiety.)

As these cyclic compounds, a six-membered ring or a five-membered ringthat contains the two carbonyl groups shown in formula I is preferable.

Of the homocyclic compounds, the preferred six-membered ring is acompound with an o-quinone shown below in general formula II: ##STR5##

(wherein R₁, R₂, R₃ and R₄ are hydrogen, hydrocarbon, polar group orpolycyclic compound moiety, independently.)

Formula II is not limited to hydrocarbons; alkyl groups, andparticularly alkyl groups with 1 to 18 carbon atoms, are preferred.Examples of the polar substitution group are carboxyl groups, carboxylicacid esters, hydroxyl groups, amino acids, mercapt groups, etc., butthey are not limited thereto. As the compound that has an o-quinonering, o-quinone and other compounds shown below in formulae III-VII canbe mentioned:

Oxidized alkyl gallate ##STR6## (wherein R₅ is alkyl.)

Partially oxidized ellagic acid ##STR7##

Completely oxidized ellagic acid ##STR8## Partially oxidized1,4-di(3,4-dihydroxyphenyl)2,3-dimethylbutane ##STR9## Completelyoxidized 1,4-di(3,4-dihydroxyphenyl)2,3-dimethylbutane ##STR10##

As a preferred example of the five membered ring compounds which belongto the homocyclic compounds, there is 1,2,3-triketohydroindene shownbelow in formula VIII. ##STR11##

As heterocyclic compounds, there are, e.g., compounds (IX) shown below.##STR12##

(wherein R₆ is hydrogen or hydrocarbon, polycyclic compound moiety; andR₇ and R₈ is hydrogen, polar group or polycyclic compound moiety,independently. The hydrocarbon and polar group are as defined in formulaII.)

As a preferred example of the compounds shown in formula (IX), there is,e.g., isatin shown below. ##STR13##

The metals that form the complex are metals other than alkaline metalsthat have o,o-ligands. The handling of complexes containing Fe, Co, Ni,Al, etc., is particularly facile, so they are preferred. The metalcomplex that is the accelerator of the activity of hydrolase of thisinvention can be obtained by the addition of a salt solution thatcontains the above-mentioned metal ions to a compound I that becomes theabove-mentioned ligand. For example, by the addition of an aqueoussolution of hydrochloride, sulfate, etc., alone or by the addition of amixture of these aqueous solutions, it is possible to obtain the metalcomplex as a reaction product. This reaction product is recovered as aprecipitate when the pH of the solution is adjusted appropriately, andit is also possible to use the reaction product in the form of asolution. For example, an oxidized propyl gallate-iron complex can beobtained from a mixture of a solution containing oxidized propyl gallatewith a solution of ferric chloride. In such a metal complex, there canbe a ligand that contains one or more kinds of halogen radicals,sulfuric acid moiety, nitric acid moiety, and ammonium moiety. Water canbe also included as a ligand.

The accelerator of blood coagulation of this invention may be furthercomprised of hydrolase as a co-accelerator The accelerator of thepresent invention increases the activity of hydrolase and may be used incombination with a hydrolase co-accelerator. This hydrolase is aprotease that can hydrolyze the bond between Arg and any amino acidresidue or the bond between Lys and any amino acid residue on thepeptide chain. As such a protease, there are, for example, serineproteases such as trypsin, thrombin, snake venom thrombin-like enzymes,etc.; thiol proteases such as cathepsin B and ficin, etc.; and metalproteases such as kinase I, etc. In particular, serine proteases aresuitable for use. These proteases used alone act to accelerate bloodcoagulation, and when used together as a co-accelerator with theaccelerator (metal complex) of the activity of hydrolase mentionedabove, the activation of blood coagulation is even more rapid.

When the hydrolase mentioned above is included, then it is included inthe proportion of 10⁻² -10⁷ parts by weight of the said enzyme (10¹-10¹⁰ units) for every 100 parts by weight of the metal complex. Even ifthere is too little enzyme included, blood coagulation will beaccelerated if there is the metal complex present, but compared to thecase when the enzyme is provided in the proportions mentioned above, theeffects are much smaller; if there is an excess, the effects comparedwith those obtained with the proportions mentioned above are notachieved.

Also, in the accelerator of blood coagulation of this invention, anorganic compound that includes amines salts and/or a quaternary nitrogencan be present. These compounds adsorb onto heparin, neutralizing it,and are used as agents to neutralize heparin. As amines constituting theamine salts, primary, secondary, and tertiary amines are all acceptable,and the acid in the structure of the amine salt can be either aninorganic acid or an organic acid. As an inorganic acid,halohydro-acids, sulfuric acid, sulfurous acid, etc., can be used, andas an organic acid, there are formic acid, acetic acid, etc. Usually,the organic residue of an amine salt is an alkyl group, but it can alsobe a hydrocarbon group including a different element such as an iminogroup, ether group, or so on. The amine salt can also be anintramolecular salt.

As a preferred example of the amine salts, for example, these arehexadecyldimethylamine hydrochloride andtetradecyldi(aminoethyl)glycine.

    C.sub.16 H.sub.33 --N.sup.+ H(CH.sub.3).sub.2 . Cl.sup.-   (XI)

    C.sub.14 H.sub.29 NHCH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 N.sup.+ H.sub.2 CH.sub.2 COO.sup.-                                        (XII)

One example of an organic compound with a quaternary nitrogen istetraalkyl ammonium. It is also acceptable for the compound to have ahydrocarbon group that contains another element such as an imino group,ether group, etc., or for the compound to have an allyl group instead ofan alkyl group. As a preferred example, there is thedodecyltrimethylammonium chloride shown in formula XIII.

    C.sub.12 H.sub.25 N.sup.+ (CH.sub.3).sub.3 . Cl .sup.-     (XIII)

In addition to these chemical compounds with a relatively low molecularweight, it is also possible to use organic polymers that have aquaternary nitrogen. As such a polymer, the polycation shown by thegeneral formula XIV with a repeating unit can be mentioned. ##STR14##

(wherein R₉ -R₁₂ is hydrogen or an alkyl group, X is a halogen group oracid moiety, and Y is an alkylene group or alkylene group-SO₂ --, andthe above-mentioned unit is repeated 5-2000 times.)

Of the compounds of formula XIV, polycations with repeated units such asthose shown in formula XV and XVI are particularly suitable. ##STR15##

When the heparin-neutralizing agent mentioned above is included, theproportions are 5-10,000 parts by weight of the said neutralizing agentto 100 parts by weight of the metal complex. If there is not enough ofthe neutralizing agent, then when the blood contains heparin, theheparin will not be neutralized and so the blood will not coagulate. Ifthere is too much of the neutralizing agent, the effects compared withthose obtained with the proportions mentioned above are not achieved.

In the accelerator of blood coagulation of this invention, ananti-fibrinolysis agent and/or an antiplasmin agent can be included.Aprotinin, soybean trypsin inhibitor, ε-aminocaproic acid,p-aminomethylbenzoic acid, 4-(aminomethyl)cyclohexanecarboxylic acid,etc., which are conventionally used clinically, can be used as theanti-fibrinolysis agent and/or the anti-plasmin agent. These can be usedindependently or in combination. For example, aprotinin is used at theproportion of about 100-600 KIU per milliliter of blood, soybean trypsininhibitor is used at the proportion of about 500-4000 FU per milliliterof blood, and ε-aminocaproic acid, p-aminomethylbenzoic acid, and4-(aminomethyl)-cyclohexanecarboxylic acid are all used at theproportion of about 10⁻² -10⁻⁸ g per milliliter of blood in theaccelerator of blood coagulation.

The accelerator of blood coagulation of this invention is used atproportions in the range of 1×10⁻¹⁰ to 1×10⁻¹ g per milliliter of blood.If the proportion is too small, the effects of acceleration of bloodcoagulation will not be obtained. If there is an excess, results willnot be attained with the proportion to the amount used.

When the accelerator of blood coagulation of this invention is used, thevessels to contain the blood for testing can be of either glass orresin. It is acceptable for the accelerator of blood coagulation to beadded to the blood after it has been collected into the vessel, and itis also acceptable for the accelerator of blood coagulation to bepresent already in the vessel to be used before blood sampling. Theaccelerator of blood coagulation can be in the form of a powder, and itcan also be dissolved, in advance, in an appropriate solvent. When theaccelerator of blood coagulation is used in the form of a powder, or asa solution of high concentration, and when one portion of the blood maycome into contact with the blood coagulation accelerator at a highconcentration, so that there is the danger that the protein of the bloodmay be denatured, the said accelerator of blood coagulation should beheld on a carrier that has a specific surface area that is large.

For the carrier that is used in this way, there are no specialrestrictions, provided that there is no harmful effect on the blood testresults and that the carrier has a large specific surface area. Forexample, unwoven cloth, textiles, resin beads, etc., are suitable foruse. To hold the accelerator of blood coagulation on such a carrier, forexample, the solution or dispersion of the accelerator can be applied tothe carrier, or the carrier can be immersed in a solution or dispersionof the accelerator, after which it is dried. Water that containsappropriate assistant agents such as gum arabic, etc., is prepared andused for a dispersion of the accelerator of blood coagulation, and thiscan be lyophilized, resulting in a accelerator of blood coagulation heldon a carrier in particulate form.

The accelerator of the activity of hydrolase of this invention is anaccelerator of the activity of enzymes that decompose proteins, andespecially an accelerator of serine protease. Serine protease has theability to cut, by hydrolysis, the bond of peptide chains between Argand any amino acid residue and also the bond between Lys and any aminoacid residue. The accelerator of the activity of hydrolase of thisinvention, when used as an accelerator of blood coagulation, firstactivates Factor XII, which is one kind of precursor of serine protease.Then, the enzyme reaction of the activated Factor XII is furtheraccelerated, and the other blood coagulation factors in the blood areactivated in turn, so that the blood coagulates in a short period oftime. When in the accelerator of blood coagulation, serine proteaseand/or other such enzymes that hydrolyze protein are present as aco-accelerator, the activation of the coagulation factors in the bloodis further accelerated. It is thought that the accelerator of theactivity of hydrolase contained in the accelerator of blood coagulationaccelerates the reaction of these enzymes that hydrolyze proteins. As aresult, the blood coagulates in a short period of time.

When an organic compound that has an amine salt or a quaternary nitrogenis contained in the accelerator of blood coagulation, this acceleratorof blood coagulation can be used to coagulate blood that containsheparin. If such an accelerator of blood is added to blood that containsheparin, the neutralizer of the amine salt, etc., adsorbs, neutralizes,and precipitates the heparin, so that the inhibition of thrombin andFactor XII by the heparin can be eliminated. For that reason, the normalability of the blood to coagulate is restored. In addition, a hydrolaseactivity accelerator (metal complex) contained in the accelerator ofblood coagulation has the ability to act on the Factor XII in the bloodto activate it. So, as when normal blood is treated, the bloodcoagulates rapidly.

When an anti-fibrinolysis agent and/or an anti-plasmin agent is alsoincluded in addition to the above-mentioned amino salts, etc., thedecomposition of fibrin by plasmin, which competes with the coagulationreaction of the blood, is inhibited. For that reason, the coagulation ofthe blood is accelerated, and in addition, after coagulation, thecoagulation is stable.

Depending on the metal complex and the variety of neutralizer containedin the accelerator of blood coagulation, the amount of accelerator ofblood coagulation, the material of the vessel used, the amount ofheparin in the blood, and the like, the time needed for bloodcoagulation varies. When the metal complex is the sole main ingredient,and if a vessel made of synthetic resin is used, the time needed isgenerally about 20-30 minutes; when a hydrolase is included as aco-accelerator, it takes about 3-7 minutes under the same conditions.When blood that contains heparin is treated, it takes about 20-40minutes for coagulation under the same conditions.

As mentioned above, it is possible by use of the accelerator of bloodcoagulation of this invention to coagulate blood that is normal and alsoblood that contains heparin in a short period of time. The blood clotsatisfactorily aggregates, and the separation effect of the serum andthe blood clot is excellent. So, when the serum is obtained, the bloodclot does not mix with the serum, and it is possible to obtain serum ina high yield.

The compound that is the main ingredient of the accelerator of bloodcoagulation of this invention is a metal complex, so compared to theorganic cyclic compound I included in the accelerator of bloodcoagulation already disclosed by the inventors, the stability to heat isstill greater. .Accordingly, even when the accelerator of bloodcoagulation of this invention undergoes such treatment as autoclavesterilization, etc., the function thereof is not reduced. It is alsopossible to store this accelerator satisfactorily for long periods oftime. When the above-mentioned organic cyclic compound is used as theaccelerator of blood coagulation, there is the danger that changes thesome kinds of serum components, but the compound included in theaccelerator of blood coagulation of this invention does not react withthe components in the blood, so that accurate test results can beobtained.

EXAMPLE 1-1

First, 50 μl of physiological saline dispersion liquid containingoxidized n-propyl gallate-iron complex (accelerator of bloodcoagulation) in a concentration of 0.1% by weight was put into acommercially available tube made of polymethylmethacrylate, and then 5ml of freshly collected human blood was added, and the tube was left at23° C. The time at which the blood clot was seen to begin to form and atwhich serum appeared was taken to be the blood coagulation time. As soonas serum appeared, the sample was put into a centrifuge and centrifugedat 1000 ×g for 5 minutes. The separation effect of the serum wasobserved by eye. The results obtained are given in Table 1. Results fromExamples 1-2 to 1-8 and Comparative Examples 1-1 and 1-2 are given inTable 1 as well.

EXAMPLE 1-2

An iron complex of oxidized ellagic acid (a compound with the structureshown in formula V) as the accelerator of blood coagulation was used.Other conditions were the same as those given for Example 1-1.

EXAMPLE 1-3

An iron complex of 1,2,3-triketohydroindene as the accelerator of bloodcoagulation was used. Other conditions were the same as those given forExample 1-1.

EXAMPLE 1-4

An iron complex of isatin as the accelerator of blood coagulation wasused. Other conditions were the same as those given for Example 1-1.

EXAMPLE 1-5

An iron complex of oxidized 1,4-di(3,4-dihydroxyphenyl)2,3-dimethylbutane as the accelerator of blood coagulation was used.Other conditions were the same as those given for Example 1-1.

EXAMPLE 1-6

A cobalt complex was used instead of an iron complex. Other conditionswere the same as those given for Example 1-2.

EXAMPLE 1-7

A nickel complex was used instead of an iron complex. Other conditionswere the same as those given for Example 1-2.

EXAMPLE 1-8

An aluminum complex was used instead of an iron complex. Otherconditions were the same as those given for Example 1-2.

COMPARATIVE EXAMPLE 1-1

The accelerator of blood coagulation was not used. Other conditions werethe same as those given for Example 1-1.

COMPARATIVE EXAMPLE 1-2

A tube made of glass was used, and the accelerator of blood coagulationwas not used. Other conditions were the same as those given for Example1-1.

                                      TABLE 1                                     __________________________________________________________________________           Accelerator of the Blood coagulation                                                                      Separation effect                                 activity of hydrolase                                                                            time (minute)                                                                          of the serum                                                                           Spitz                             __________________________________________________________________________    Example 1-1                                                                          Oxidized n-propyl gallate-iron complex                                                           30       Excellent                                                                              Polymethyl                                                                    methacrylate                      Example 1-2                                                                          Oxidized ellagic acid-iron complex                                                               25       Excellent                                                                              Polymethyl                                                                    methacrylate                      Example 1-3                                                                          1.2.3-triketohydroindene-iron complex                                                            30       Excellent                                                                              Polymethyl                                                                    methacrylate                      Example 1-4                                                                          Isatin-iron complex                                                                              25       Excellent                                                                              Polymethyl                                                                    methacrylate                      Example 1-5                                                                          Oxidized 1.4-di(3.4-dihydroxyphenyl)                                                             30       Excellent                                                                              Polymethyl                               2.3-dimethylbutane-iron complex      methacrylate                      Example 1-6                                                                          Oxidized ellagic acid-coball complex                                                             25       Excellent                                                                              Polymethyl                                                                    methacrylate                      Example 1-7                                                                          Oxidized ellagic acid-nickel complex                                                             25       Excellent                                                                              Polymethyl                                                                    methacrylate                      Example 1-8                                                                          Oxidized ellagic acid aluminum                                                                   25       Excellent                                                                              Polymethyl                               complex                              methacrylate                      Comparative                                                                          --                 260      Precipitate of                                                                         Polymethyl                        Example 1-1                        fibrin in the serum                                                                    methacrylate                      Comparative                                                                          --                 65       Precipitate of                                                                         Glass                             Example 1-2                        fibrin in the serum                        __________________________________________________________________________

EXAMPLE 2 (1-1)

A physiological saline solution dispersion liquid, which contained aniron complex of oxidized ellagic acid (a compound with the structureshown in formula V) as an agent to activate blood coagulation factors(the metal complex), and trypsin as a co-accelerator of bloodcoagulation in concentrations of 0.5% by weight and 0.05% by weight,respectively, was prepared. Freshly collected human blood (3 ml) was putinto a commercially available plain tube made of polyethylene, and 30 μlof the solution of the accelerator of blood coagulation described abovewas added thereto. The plain tube was left at room temperature, and thetime needed for the blood to lose its ability to flow was measured, andtaken to be the blood coagulation time. Then, the sample was centrifugedafter coagulation at 3000 rpm for 5 minutes, and the separation effectof the serum was evaluated by eye. The results are shown in Table 2.Results for Examples 2 (1-2) to 2 (4-3) are also given in Table 2.

EXAMPLE 2 (1-2)

Thrombin was used as a hydrolase of a co-accelerator, and aphysiological saline solution dispersion liquid of the accelerator ofblood coagulation was prepared so that the concentration of the saidenzyme was 500 units/ml. Other conditions were the same as in Example 2(1-1).

EXAMPLE 2 (1-3)

Snake venom thrombin-like enzyme was used as a hydrolase of aco-accelerator, and a physiological saline solution dispersion liquid ofthe accelerator of blood coagulation was prepared so that theconcentration of the said enzyme was 0.005% by weight. Other conditionswere the same as in Example 2 (1-1).

EXAMPLE 2 (2-1)

An iron complex of 1,2,3-triketohydroindene as a metallic complex wasused. Other conditions were the same as those given for Example 2 (1-1).

EXAMPLE 2 (2-2)

An iron complex of 1,2,3-triketohydroindene as a metallic complex wasused. Other conditions were the same as those given for Example 2 (1-2).

EXAMPLE 2 (2-3)

An iron complex of 1,2,3-triketohydroindene as a metallic complex wasused. Other conditions were the same as those given for Example 2 (1-3).

EXAMPLE 2 (3-1)

An iron complex of oxidized n-propyl gallate as a metallic complex wasused. Other conditions were the same as those given for Example 2 (1-1).

EXAMPLE 2 (3-2)

An iron complex of oxidized n-propyl gallate as a metallic complex wasused. Other conditions were the same as those given for Example 2 (1-2).

EXAMPLE 2 (3-3)

An iron complex of oxidized n-propyl gallate as a metallic complex wasused. Other conditions were the same as those given for Example 2 (1-3).

EXAMPLE 2 (4-1)

An iron complex of isatin as a metallic complex was used. Otherconditions were the same as those given for Example 2 (1-1). EXAMPLE 2(4-2)

An iron complex of isatin as a metallic complex was used. Otherconditions were the same as those given for Example 2 (1-2).

EXAMPLE 2 (4-3)

An iron complex of isatin as a metallic complex was used. Otherconditions were the same as those given for Example 2 (1-3).

EXAMPLE 2 (5-1)

A cobalt complex of oxidized ellagic acid (a compound with the structureshown in formula V) as a metallic complex was used. Other conditionswere the same as those given for Example 2 (1-1). The results obtainedare given in Table 3. Results from Examples 2 (5-2) to 2 (7-3) are givenin Table 3 as well.

EXAMPLE 2 (5-2)

A cobalt complex of oxidized ellagic acid (a compound with the structureshown in formula V) as a metallic complex was used. Other conditionswere the same as those given for Example 2 (1-2).

EXAMPLE 2 (5-3)

A cobalt complex of oxidized ellagic acid (a compound with the structureshown in formula V) as a metallic complex was used. Other conditionswere the same as those given for Example 2 (1-3).

EXAMPLE 2 (6-1)

A nickel complex of oxidized ellagic acid (a compound with the structureshown in formula V) as a metallic complex was used. Other conditionswere the same as those given for Example 2 (1-1).

EXAMPLE 2 (6-2)

A nickel complex of oxidized ellagic acid (a compound with the structureshown in formula V) as a metallic complex was used. Other conditionswere the same as those given for Example 2 (1-2).

EXAMPLE 2 (6-3)

A nickel complex of oxidized ellagic acid (a compound with the structureshown in formula V) as a metallic complex was used. Other conditionswere the same as those given for Example 2 (1-3).

EXAMPLE 2 (7-1)

An aluminum complex of oxidized ellagic acid (a compound with thestructure shown in formula V) as a metallic complex was used. Otherconditions were the same as those given for Example 2 (1-1).

EXAMPLE 2 (7-2)

An aluminum complex of oxidized ellagic acid (a compound with thestructure shown in formula V) as a metallic complex was used. Otherconditions were the same as those given for Example 2 (1-2).

EXAMPLE 2 (7-3)

An aluminum complex of oxidized ellagic acid (a compound with thestructure shown in formula V) as a metallic complex was used. Otherconditions were the same as those given for Example 2 (1-3).

Comparative Example 2-1

Trypsin was used alone as the accelerator of blood coagulation. Otherconditions were the same as given for Example 2 (1-1).

COMPARATIVE EXAMPLE 2-2

Thrombin was used alone as the accelerator of blood coagulation. Otherconditions were the same as given for Example 2 (1-2).

COMPARATIVE EXAMPLE 2-3

Snake venom thrombin-like enzymes were used alone as the accelerator ofblood coagulation. Other conditions were the same as given for Example 2(1-3).

COMPARATIVE EXAMPLE 2-4

The accelerator of blood coagulation was not used. Other conditions werethe same as those given for Example 2 (1-1).

COMPARATIVE EXAMPLE 2-5

The accelerator of blood coagulation was not used, and a glass Spitz wasused. Other conditions were the same as those given for Example 2 (1-1).

                                      TABLE 2                                     __________________________________________________________________________                                     Blood coagu-                                                                         Separation                                                             lation time                                                                          effect of                                     Metallic complex                                                                           Hydrolase   (minute)                                                                             the serum                                                                           Spitz                           __________________________________________________________________________    Example 2 (1-1)                                                                       Oxidized ellagic acid(V)-                                                                  Trypsin     5      Excellent                                                                           Polyethylene                            iron complex                                                          Example 2 (1-2)                                                                       Oxidized ellagic acid(V)-                                                                  Thrombin    3      Excellent                                                                           Polyethylene                            iron complex                                                          Example 2 (1-3)                                                                       Oxidized ellagic acid(V)-                                                                  Snake venom thrombin-                                                                     5      Excellent                                                                           Polyethylene                            iron complex like enzymes                                             Example 2 (2-1)                                                                       1.2.3-triketohydroindene-                                                                  Trypsin     5      Excellent                                                                           Polyethylene                            iron complex                                                          Example 2 (2-2)                                                                       1.2.3-triketohydroindene-                                                                  Thrombin    4      Excellent                                                                           Polyethylene                            iron complex                                                          Example 2 (2-3)                                                                       1.2.3-triketohydroindene-                                                                  Snake venom thrombin-                                                                     4      Excellent                                                                           Polyethylene                            iron complex like enzymes                                             Example 2 (3-1)                                                                       Oxidized n-propyl                                                                          Trypsin     6      Excellent                                                                           Polyethylene                            gallate-iron complex                                                  Example 2 (3-2)                                                                       Oxidized n-propyl                                                                          Thrombin    4      Excellent                                                                           Polyethylene                            gallate-iron complex                                                  Example 2 (3-3)                                                                       Oxidized n-propyl                                                                          Snake venom thrombin-                                                                     5      Excellent                                                                           Polyethylene                            gallate-iron complex                                                                       like enzymes                                             Example 2 (4-1)                                                                       Isatin-iron complex                                                                        Trypsin     7      Excellent                                                                           Polyethylene                    Example 2 (4-2)                                                                       Isatin-iron complex                                                                        Thrombin    3      Excellent                                                                           Polyethylene                    Example 2 (4-3)                                                                       Isatin-iron complex                                                                        Snake venom thrombin-                                                                     5      Excellent                                                                           Polyethylene                                         like enzymes                                             __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________                                     Blood coagu-                                                                         Separation                                                             lation time                                                                          effect of                                     Metallic complex                                                                           Hydrolase   (minute)                                                                             the serum                                                                           Spitz                           __________________________________________________________________________    Example 2 (5-1)                                                                       Oxidized ellagic acid(V)-                                                                  Trypsin     5      Excellent                                                                           Polyethylene                            cobalt complex                                                        Example 2 (5-2)                                                                       Oxidized ellagic acid(V)-                                                                  Thrombin    4      Excellent                                                                           Polyethylene                            cobalt complex                                                        Example 2 (5-3)                                                                       Oxidized ellagic acid(V)-                                                                  Snake venom thrombin-                                                                     5      Excellent                                                                           Polyethylene                            cobalt complex                                                                             like enzymes                                             Example 2 (6-1)                                                                       Oxidized ellagic acid(V)-                                                                  Trypsin     5      Excellent                                                                           Polyethylene                            nickel complex                                                        Example 2 (6-2)                                                                       Oxidized ellagic acid(V)-                                                                  Thrombin    5      Excellent                                                                           Polyethylene                            nickel complex                                                        Example 2 (6-3)                                                                       Oxidized ellagic acid(V)-                                                                  Snake venom thrombin-                                                                     4      Excellent                                                                           Polyethylene                            nickel complex                                                                             like enzymes                                             Example 2 (7-1)                                                                       Oxidized ellagic acid(V)-                                                                  Trypsin     5      Excellent                                                                           Polyethylene                            aluminum complex                                                      Example 2 (7-2)                                                                       Oxidized ellagic acid(V)-                                                                  Thrombin    4      Excellent                                                                           Polyethylene                            aluminum complex                                                      Example 2 (7-3)                                                                       Oxidized ellagic acid(V)-                                                                  Snake venom thrombin-                                                                     5      Excellent                                                                           Polyethylene                            aluminum complex                                                                           like enzymes                                             __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________                                Blood coagulation                                                                      Separation effect                               Metallic complex                                                                       Hydrolase   time (minute)                                                                          of the serum                                                                             Spitz                         __________________________________________________________________________    Comparative                                                                          --       Trypsin     14       Excellent  Polyethylene                  Example 2-1                                                                   Comparative                                                                          --       Thrombin    14       Excellent  Polyethylene                  Example 2-2                                                                   Comparative                                                                          --       Snake venom thrombin-                                                                     40       Free blood cells seen                                                                    Polyethylene                  Example 2-3     like enzymes                                                  Comparative                                                                          --       --          330      Supernatant contained                                                                    Polyethylene                  Example 2-4                          fibrin                                   Comparative                                                                          --       --          60       Supernatant contained                                                                    Glass                         Example 2-5                          much fibrin                              __________________________________________________________________________

EXAMPLE 3-1

First, an aqueous dispersion liquid containing an iron complex ofoxidized ellagic acid (a compound with the structure of formula V) andpolycation (a compound with the structure of formula XVI) was prepared.A non-woven cloth of polyacetate was then impregnated with thedispersion, and dried thoroughly. There was 10⁻⁴ g of each of the abovetwo components contained on each square centimeter of unwoven cloth.

Then, 8 ml of freshly collected human blood that contained heparin atthe concentration of 2 units/ml was injected into a commerciallyavailable 10-ml tube made of polyethylene, and 1 cm² of the unwovencloth carrying the above-mentioned ingredients thereon was added andgently agitated before being left at 20° C. The time needed for thewhole blood to lose its ability to flow was taken to be the bloodcoagulation time.

As soon as the blood had coagulated, it was centrifuged for 5 minutes atthe rate of 3000 rpm. Then the separation effect of the serum wasevaluated by eye. The serum was obtained by the use of a pipette, andits volume was taken to be the yield of serum. These results are shownin Table 5. Results of Examples 3-2 to 3-10 and of Comparative Example 3are also given in Table 5.

EXAMPLE 3-2

A physiological saline solution dispersion liquid containing oxidizedn-propyl gallate-iron complex and tetradecyldi(aminoethyl)glycine inconcentrations of 0.5% and 0.2% by weight, respectively, was prepared.

In a 10-ml commercially available tube made of polyethylene, 8 ml offreshly collected human blood that contained 2 units of heparin permilliliter was injected, and then 80 μl of the above-mentioneddispersion liquid was added thereto. Other treatment was the same as inExample 3-1, and the results were evaluated.

EXAMPLE 3-3

First, 1 g of the iron complex isatin, 0.4 g of hexadecyldimethylaminehydrochloride, and 1 kg of polystyrene beads with the mean diameter of1.5 mm. as a carrier were mixed well with a small volume of ethanol asthe assistant agent for dispersal. The mixture was then dried. In onegram of the said particulate holding thereon the accelerator of bloodcoagulation, there was 10⁻³ g of iron complex of isatin and 0.4×10⁻³ gof hexadecyldimethylamine hydrochloride.

In a 10-ml commercially available tube made of polyethylene, 8 ml offreshly collected human blood that contained 2 units of heparin permilliliter was injected, and then 1 g of the above-mentioned particulateholding thereon the accelerator of blood coagulation was added thereto.The sample was treated as for Example 3-1, and then evaluated.

EXAMPLE 3-4

An iron complex of o-quinone and polycation (a compound with thestructure shown in formula XV) were used instead of the iron complex ofoxidized n-propyl gallate and tetradecyldi(aminoethyl)glycine,respectively. The iron complex of o-quinone and polycation were employedin concentration of 0.5% by weight and 0.4% by weight, respectively.Other conditions were the same as those given for Example 3-2.

EXAMPLE 3-5

An iron complex of 1,2,3-triketohydroindene and polycation (a compoundwith the structure shown in formula XVI) were used instead of the ironcomplex of oxidized n-propyl gallate andtetradecyldi(aminoethyl)glycine, respectively. Other conditions were thesame as those given for Example 3-2.

EXAMPLE 3-6

Dodecyltrimethylammonium chloride were used instead of thetetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 3-2.

EXAMPLE 3-7

An iron complex of oxidized1,4-di(3,4-dihydroxyphenyl)2,3-dimethylbutane and polycation (andpolycation (a compound with the structure shown in formula XV) were usedinstead of the iron complex of oxidized n-propyl gallate andtetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 3-2.

EXAMPLE 3-8

A cobalt complex of oxidized ellagic acid (V) and polycation (a compoundwith the structure shown in formula XV) were used instead of the ironcomplex of oxidized n-propyl gallate andtetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 3-2.

EXAMPLE 3-9

A nickel complex of ellagic acid (V) and polycation (a compound with thestructure shown in formula XV) were used instead of the iron complex ofoxidized n-propyl gallate and tetradecyldi(aminoethyl)glycine. Otherconditions were the same as those given for Example 3-2.

EXAMPLE 3-10

An aluminum complex of ellagic acid (V) and polycation (a compound withthe structure shown in formula XV) were used instead of the iron complexof oxidized n-propyl gallate and tetradecyldi(aminoethyl)glycine. Otherconditions were the same as those given for Example 3-2.

COMPARATIVE EXAMPLE 3

In a 10-ml commercially available tube made of polyethylene, 8 ml offreshly collected human blood that contained 2 units of heparin permilliliter was injected, and then without the addition of theaccelerator of blood coagulation, the same treatment as for Example 3-1was carried out, and the results were evaluated.

                                      TABLE 5                                     __________________________________________________________________________                                               Separation                                Accelerator of the activity of hydrolase                                                                 Blood coagulation                                                                      effect of                                                                            Yield of serum                     Metallic complex                                                                           Neutralizing-agent                                                                          time (minute)                                                                          the serum                                                                            (ml)                        __________________________________________________________________________    Example 3-1                                                                          Oxidized ellagic acid (V)-                                                                 Polycation (XVI)                                                                            35       Excellent                                                                            4.0                                iron complex                                                           Example 3-2                                                                          Oxidized n-propyl gallate-                                                                 Tetradocyldi(aminoethyl)                                                                    35       Excellent                                                                            4.1                                iron complex glycine                                                   Example 3-3                                                                          Isatin-iron complex                                                                        Hexadecyldimethylamine                                                                      35       Excellent                                                                            4.1                                             hydrochloide                                              Example 3-4                                                                          o-quinone-iron complex                                                                     Polycation (XV)                                                                             35       Excellent                                                                            4.0                         Example 3-5                                                                          1.2.3-triketohydroindene-                                                                  Polycation (XVI)                                                                            40       Excellent                                                                            4.1                                iron complex                                                           Example 3-6                                                                          Oxidized n-propyl gallate-                                                                 Dodecyltrimethylammonium                                                                    35       Excellent                                                                            4.1                                iron complex chloride                                                  Example 3-7                                                                          Oxidized 1.4-di(3.4-                                                                       Polycation (XV)                                                                             40       Excellent                                                                            4.1                                dihydroxylphenyl(2.3-                                                         dimethylbutane-iron                                                           complex                                                                Example 3-8                                                                          Oxidized ellagic acid (V)-                                                                 Polycation (XV)                                                                             35       Excellent                                                                            4.1                                cobalt complex                                                         Example 3-9                                                                          Oxidized ellagic acid (V)-                                                                 Polycation (XV)                                                                             35       Excellent                                                                            4.0                                nickel complex                                                         Example 3-10                                                                         Oxidized ellagic acid (V)-                                                                 Polycation (XV)                                                                             40       Excellent                                                                            4.0                                aluminum complex                                                       Comparative                                                                          --           --            Not coagulated                                                                         Separation of                                                                        --                          Example 3                                  the plasma                         __________________________________________________________________________

EXAMPLE 4-1

Polyester unwoven cloth was impregnated with an aqueous dispersionliquid containing an iron complex of oxidized ellagic acid (a compoundwith the structure shown in formula V), polycation (a compound with thestructure shown in formula XVI), and aprotinin, and was then completelydried. There were, per square centimeter of unwoven cloth, 4×10⁻⁴ g ofthe iron complex, 4×10⁻⁴ g of polycation, and 500 KIU of aprotinin.

In a 5-ml commercially available tube made of polyethylene, 2 ml offreshly collected human blood that contained 1.0 IU of heparin permilliliter was injected, and then 1 cm² of the unwoven cloth holdingthereon the ingredients mentioned above was added thereto, gentlyagitated, and then left at 20 ° C. One hour later, or 30 hours later,the serum was collected, and the fibrin and fibrinogen degradationproduct (FDP) were assayed. The results are given in Table 6. There wasno difference in the results of assays of FDP after 1 hour or 30 hours,which shows that the decomposition reaction of the blood clot wasinhibited. Results for Examples 4-2 to 4-10 and of Comparative Example 4are also given in Table 6.

EXAMPLE 4-2

A dispersion in physiological saline solution was made so that theconcentration of oxidized n-propyl gallate was 0.5 % by weight, that oftetradecyldi(aminoethyl)glycine was 0.5% by weight, and that ofaprotinin was 10,000 KIU/ml.

In a 5-ml commercially available tube made of polyethylene, 2 ml offreshly collected human blood that contained 1.0 IU of heparin wasinjected, and then 50 μl of the dispersion liquid mentioned above wasadded thereto. Then, the sample was treated as in Example 4-1, and theresults were evaluated.

EXAMPLE 4-3

First, 1 g of isatin, 0.4 g of hexadecyldimethylamine hydrochloride, 50mg of 4-(aminomethyl)cyclohexanecarboxylic acid, and 1 kg of polystyrenebeads with the mean diameter of 1.5 mm as a carrier were mixedthoroughly with a small amount of ethanol as assistant agent fordispersal, and dried.

In a 5-ml commercially available tube made of polyethylene, 2 ml offreshly collected human blood that contained 1.0 IU of heparin permilliliter was injected. Then, 0.3 g of the accelerator of bloodcoagulation described above was added thereto. Then, the sample wastreated as in Example 4-1, and the results were evaluated.

EXAMPLE 4-4

An iron complex of o-quinone, a polycation (a compound with thestructure shown in formula XV) and ε-aminocaproic acid were used insteadof the iron complex of oxidized n-propyl gallate,tetradecyl(aminoethyl)glycine and aprotinin. The iron complex ofo-quinone, the polycation and the ε-aminocaproic acid were employed inconcentrations of 0.5% by weight, 0.5% by weight and 0.1% by weight,respectively. Other conditions were the same as those given for Example4-2.

EXAMPLE 4-5

An iron complex of 1,2,3-triketohydroindene, a polycation (a compoundwith the structure shown in formula XVI) and ε-aminocaproic acid wereused instead of the iron complex of oxidized n-propyl gallate,tetradecyl(aminoethyl)glycine and aprotinin. Other conditions were thesame as those given for Example 4-2.

EXAMPLE 4-6

Dodecyltrimethylammonium chloride was used instead of thetetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 4-2.

EXAMPLE 4-7

An oxidized iron complex of 1,4-di(3,4-dihydroxyphenyl)2,3-dimethylbutane and polycation (a compound with the structure shownin formula XV) were used instead of the iron complex of oxidizedn-propyl gallate and tetradecyldi(aminoethyl)glycine. Other conditionswere the same as those given for Example 4-2.

EXAMPLE 4-8

A cobalt complex of oxidized ellagic acid (V) and polycation (a compoundwith the structure shown in formula XV) were used instead of the ironcomplex of oxidized n-propyl gallate andtetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 4-2.

EXAMPLE 4-9

A nickel complex of oxidized ellagic acid (V) and polycation (a compoundwith the structure shown in formula XV) were used instead of the ironcomplex of oxidized n-propyl gallate andtetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 4-2.

EXAMPLE 4-10

An aluminium complex of oxidized ellagic acid (V) and polycation (acompound with the structure shown in formula XV) were used instead ofthe iron complex of oxidized n-propyl gallate andtetradecyldi(aminoethyl)glycine. Other conditions were the same as thosegiven for Example 4-2.

                                      TABLE 6                                     __________________________________________________________________________                                                      Results of assays of                                                          FDP                                 Accelerator of the activity of hydrolase  After After                         Metallic complex                                                                            Neutralizing-agent                                                                          Anti-fibrinolysis agent                                                                     one hour                                                                            thirty                __________________________________________________________________________                                                            hours                 Example 4-1                                                                           Oxidized ellagic acid (V)-                                                                  Polycation (XVI)                                                                            Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  iron complex                              or less                                                                             or less               Example 4-2                                                                           Oxidized n-propyl gallate-                                                                  Tetradocyldi(aminoethyl)                                                                    Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  iron complex  glycine                     or less                                                                             or less               Example 4-3                                                                           Isatin-iron complex                                                                         Hexadecyldimethylamine                                                                      Aminomethylcyclohexane                                                                      2.5 μg/ml                                                                        2.5 μg/ml                                hydrochloide  carboxylic acid                                                                             or less                                                                             or less               Example 4-4                                                                           o-quinone-iron complex                                                                      Polycation (XV)                                                                             ε-aminocaproic                                                                      2.5 μg/ml                                                                        2.5 μg/ml                                                            or less                                                                             or less               Example 4-5                                                                           1.2.3-triketohydroindene-                                                                   Polycation (XVI)                                                                            ε-aminocaproic                                                                      2.5 μg/ml                                                                        2.5 μg/ml                  iron complex                              or less                                                                             or less               Example 4-6                                                                           Oxidized n-propyl gallate-                                                                  Dodecyltrimethylammonium                                                                    Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  iron complex  chloride                    or less                                                                             or less               Example 4-7                                                                           Oxidized 1.4-di(3.4-                                                                        Polycation (XV)                                                                             Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  dihydroxylphenyl(2.3-                     or less                                                                             or less                       dimethylbutane-iron                                                           complex                                                               Example 4-8                                                                           Oxidized ellagic acid (V)-                                                                  Polycation (XV)                                                                             Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  cobalt complex                            or less                                                                             or less               Example 4-9                                                                           Oxidized ellagic acid (V)-                                                                  Polycation (XV)                                                                             Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  nickel complex                            or less                                                                             or less               Example 4-10                                                                          Oxidized ellagic acid (V)-                                                                  Polycation (XV)                                                                             Aprotinin     2.5 μg/ml                                                                        2.5 μg/ml                  aluminum complex                          or less                                                                             or                    __________________________________________________________________________                                                            less              

EXAMPLE 4-11

All of the accelerators of blood coagulation described in Examples 4-1to 4-10 were prepared. Each accelerator was placed into a tube for bloodcollection, and then freshly collected human blood that containedheparin was injected into the tube. The tubes were gently agitated, andleft at 20° C. Then the samples were timed for the time needed for thewhole blood to lose its ability to flow. That is, the blood coagulationtime was measured. In all of the examples, blood coagulated in 35-40minutes. After the blood had coagulated, the samples were immediatelycentrifuged at the speed of 3000 rpm for 5 minutes, and the separationeffect of the serum was evaluated by eye. The collection of the serum bypipette was also studied. In all examples, the separation effect ofserum and the yield of serum were excellent.

EXAMPLE 5-1

First, 50 -1 of physiological saline solution dispersion liquidcontaining an iron complex of oxidized ellagic acid (a compound with thestructure shown in formula V) in a concentration of 0.1% by weight wasput into a commercially available tube made of polymethylmethacrylate,and then 5 ml of freshly collected human blood was added thereto, andthe tube was left at 23 ° C. After the completion of blood coagulation,the sample was centrifuged at 1000×g for 5 minutes so as to separate theserum from the blood clot. Then, uric acid (UA), phospholipids (PL) andtriglycerides (TG) that are present in the separated serum weremeasured, the results of which are shown in Table 7. The results fromComparative Examples 5-1 and 5-2 mentioned below are shown in Table 7 aswell.

Comparative Example 5-1

This example is the same as Example 5-1 except that oxidized ellagicacid shown in formula V was used as an accelerator of blood coagulation.

COMPARATIVE EXAMPLE 5-2

This example is the same as Example 5-1 except that the dispersionliquid of the accelerator of blood coagulation was not used.

According to the comparison with Comparative Example 5-2, Table 7indicates that UA, PL, and TG can be measured with accuracy in the serumobtained from Example 5-1 in which metal complex was used as anaccelerator of blood coagulation, whereas the contents of UA, PL, and TGin the serum obtained from Comparative Example 5-1 in which oxidizedellagic acid that does not form a metal complex was used cannot beaccurately measured (namely, the contents of UA, PL, and TG in the serumof this comparative example are lower than those of UA, PL, and TG inthe serum from Comparative Example 5-2). These facts mean that theaccelerator of blood coagulation of Comparative EXAMPLE 5-1 influencesthe assay of the contents of the above-mentioned components in the serumfor the worse.

                  TABLE 7                                                         ______________________________________                                                     UA      PL        TG                                                          (mg/dl) (mg/dl)   (mg/dl)                                        ______________________________________                                        Example 5-1    6.1       155       90                                         Comparative Example 5-1                                                                      5.0       130       64                                         Comparative Example 5-2                                                                      6.1       156       93                                         ______________________________________                                    

EXAMPLE 6

First, 5 ml of an aqueous dispersion liquid containing an iron complex(an accelerator of blood coagulation) of oxidized ellagic acid (acompound represented by formula V) in a concentration of 0.1% by weightwas put into a hard glass tube, which was then heat-treated at 121° C.for 1 hour under two atmospheres of pressure. The said accelerator ofblood coagulation was not denatured at all before and after suchautoclave treatment.

COMPARATIVE EXAMPLE 6

First, 5 ml of an aqueous dispersion liquid containing oxidized ellagicacid (a compound represented by formula V) in a concentration of 0.1% byweight was put into a hard glass tube, which was then autoclaved in thesame manner as that of Example 6. The oxidized ellagic acid, which wasblackish-brown before the said autoclave treatment, becameyellowish-white after the autoclave treatment. Moreover, the oxidebecame water-soluble, which means that the oxide was denatured.

It is understood that various other modifications will be apparent toand can be readily made by those skilled in the art without departingfrom the scope and spirit of this invention. Accordingly, it is notintended that the scope of the claims appended hereto be limited to thedescription as set forth herein, but rather that the claims be construedas encompassing all the features of patentable novelty that reside inthe present invention, including all features that would be treated asequivalents thereof by those skilled in the art to which this inventionpertains.

What is claimed is:
 1. An accelerator of the activity of hydrolase,comprising an iron complex containing the oxidized ellagic acid ofStructure V as a ligand. ##STR16## ##STR17##
 2. An accelerator accordingto claim 1, wherein said hydrolase is serine protease.
 3. An acceleratoraccording to claim 2, wherein said serine protease is blood coagulationFactor XII.
 4. The accelerator of claim 1, wherein said iron complexaccelerates blood coagulation.
 5. The accelerator of claim 2, whereinsaid iron complex accelerates blood coagulation.
 6. The accelerator ofclaim 3, wherein said iron complex accelerates blood coagulation.